1. Field of the Invention
This invention relates to opioid receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian opioid receptor gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel opioid receptor gene, said recombinant expression constructs being capable of expressing opioid receptor protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel opioid receptor protein. The invention also provides cultures of such cells producing this opioid receptor protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel opioid receptor protein are also provided by the invention.
2. Background of the Invention
The use (and abuse) of opiates, archetypally opium and morphine, have been known since antiquity (reviewed in Brownstein, 1993, Proc. Natl. Acad. Sci. USA 90: 5391-5393). Since the nineteenth century, chemical characterization and synthesis of a number of morphine analogues have been achieved in an effort to discover a compound with the analgesic effects of morphine that lacks or is substantially attenuated in its addictive potential. These efforts have proven fruitless to date.
The biology behind the reasons why morphine and morphine-like compounds display both analgesic and addictive properties was first elucidated by the discovery of endogenous morphine-like compounds termed enkephalins (see DiChara & North, 1992, Trends in Phamacol. Sci. 13: 185-193 for review). Accompanying this finding of an endogenous opiate was the biochemical evidence for a family of related but distinct opiate receptors, each of which displays a unique pharmacological profile of response to opiate agonists and antagonists (see McKnight & Rees, 1991, Neurotransmissions 7: 1-6 for review). To date, four distinct opiate receptors have been described by their pharmacological profiles and anatomical distribution; these comprise the .mu., .delta., .kappa. and .sigma. receptors (the .sigma. receptor has been determined to be a non-opioid receptor with cross-reactivity to some opioid agonists).
Thus, mammalian opioid receptors are known in the art, and some of these proteins have been isolated biochemically and their corresponding genes have been recently cloned using genetic engineering means.
Kieffer et al., 1992, Proc. Natl. Acad. Sci. USA 89: 12048-12052 disclosed the isolation of a cDNA copy of the mouse .delta.-opioid receptor by expression cloning.
Evans et al., 1992, Science 258: 1952-1955 disclose the isolation of a cDNA copy of the mouse .delta.-opioid receptor by expression cloning.
Chen et al., 1993, Molec. Pharmacol. 44: 8-12 disclose the isolation of a cDNA copy of the rat .mu.-opioid receptor.
Yasuda et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6736-6740 disclose the isolation of a cDNA copy of each of the mouse .kappa.- and .delta.-opioid receptor.
Bzdega et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9305-9309 disclose the isolation and chromosomal location of the .delta.-opioid receptor in the mouse.
In 1991, U.S. pharmaceutical companies spent an estimated $7.9 billion on research and development devoted to identifying new therapeutic agents (Pharmaceutical Manufacturer's Association). The magnitude of this amount is due, in part, to the fact that hundreds, if not thousands, of chemical compounds must be tested in order to identify a single effective therapeutic agent that does not engender unacceptable levels of undesirable or deleterious side effects. There is an increasing need for economical methods of testing large number of chemical compounds to quickly identify those compounds that are likely to be effective in treating disease.
This is of particular importance for psychoactive and psychotropic drugs, due to their pharmacological importance and their potential to greatly benefit or greatly harm human patients treated with such drugs. At present, few such economical systems exist. Conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-receptor) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. The ability to synthesize human opioid receptor molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds. For these and other reasons, development in vitro screening methods for psychotropic drugs has numerous advantages and is a major research goal in the pharmaceutical industry.